A bitopic agonist bound to the dopamine 3 receptor reveals a selectivity site

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity and improve current medications by reducing off-target side effects. However, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R:GαOβγ complex bound to the D3R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.


Potencies
of the D3R:GO:FOB02-04A complex and validation of the L 3.41 W mutation.A Size exclusion chromatogram of the D3R:G O :FOB02-04A sample (left).SDS-PAGE of the pure D3R:G O :FOB02-04A sample with superposed in-gel fluorescence for the GFP-D3R (right).B Concentration-response curves of D3R WT (blue) compared to L 3.41 W variant (orange) upon G OA activation with FOB02-04A using the TRUPATH assay.Data are presented as means ± SEM of five (D 3 R WT) and six (L 3.41 W) independent experiments performed in technical triplicate.Source data are provided as a Source Data fileSupplementary Figure 2. Cryo-EM of the single particle reconstruction of the D 3 R:G O :FOB02-04A complex structure.A Flow chart of data processing.B Representative micrograph (0.84 Å/ pixel) of the D 3 R:G O :FOB02-04A dataset collected using a Titan Krios with a K3 detector.C Representative 2D class averages of the D 3 R:G O :FOB02-04A complex.D-E FSC curve of the final reconstruction showing an overall resolution of 3.05 Å and 3.09 Å for Conformation A (D) and B (E). F-G Local resolution estimation of the D 3 R:G O :FOB02-04A map as calculated by CryoSPARC for Conformation A (F) and Conformation B (G). H-I Angular distribution of all particles used in the final reconstruction for Conformation A (of centering the ligand binding site on the cryo-EM box.A Slices from 3D refinement before and after re-centering the particles at the OBS.B Local resolution of the cryo-EM maps (as calculated by CryoSPARC) with an inset into the ligand binding site before and after OBS re-centering.The center of the box is marked with a green dot in the 2D slices and 3D maps.-EM map quality and model fit of the D3R:G O :FOB02-04A complexes.Protein shown as cartoons with residues represented as sticks and colored by subunit (yellow D3R and cyan Gα O ) or ligand (dark red for Conformation A and green for Conformation B).Cryo-EM density is shown as mesh with color corresponding to each component.A-C Transmembrane helices of D3R Conformation A, the α5-helix of G O and FOB02-04A in Conformation A. D-F Transmembrane helices of D3R Conformation B, the α5-helix of G O and FOB02-04A in Conformation B. of the D3R by FOB02-04A.A Two orthogonal views of the D3R-FOB02-04A (yellow, cartoons) superposed to the inactive state eticlopride-bound (PDB 3PBL, cyan cartoons) and active state pramipexole-bound D3R (PDB 7CMU, orange cartoons).Conserved motifs are highlighted with relevant residues displayed as sticks.B Overall view of G o selected mutations to G i equivalent residues (highlighted in violet) (top panel) and concentration-response curves of D 3 R upon G OA mutants activation by FOB02-04A using TRUPATH assay (shown as net BRET, bottom panel).Data are derived from three (G OA I28 G.HN.52 E, N194 G.s2s3.02 D, Y354 G.H5.26 F) and four (G OA -V334 G.H5.06 F, G350 G.H5.22 D) independent experiments performed in technical triplicate.Source data are provided as a Source Data File.simulations of D 3 R:Gα O βγ interactions with bitopic FOB02-04A and pramipexole.A-B Salt bridge interaction between negatively charged carboxyl group of polar residue D341 G.H5.13 of Gα O C-terminal α5 and positively charged guanidinium group of FOB02-04A:D 3 R polar residue R218 5.68 (A) and polar residue R222 5.72 (B).C Closest distance between Q139 34.54 in 3 R and terminal tertiary amine moeiety of K32 G.hsn1.03 of Gα O N-terminal helix.D-E Salt bridge interaction between oxygen atoms of carboxyl group of D110 3.32 in D 3 R with the basic nitrogen of trans-cyclopropyl amine linkage of FOB02-04A (D) and with the atom N1 of the propylamino group of pramipexole (E).F Hydrogen bond interaction between oxygen atom S196 5.46 in D 3 R with the atom type bitopic FOB0204-A within the D 3 R:Gα O βγ complex.G Frequency of interactions indicates the presence of two distinct conformational states of estimated distribution of 90% for Conformation A and 10% for Conformation B. H-I Closest distances between oxygen atoms of carboxyl group of E90 2.65 in D 3 R with the atom type N5 of the indole moiety (H) and atom N4 of the amide group of FOB02-04A (I).Data from five independent simulations of D 3 R:Gα O βγ heterotrimer complex are shown, spanning 0.6 μs of cumulative time per system, with the sampling rate of 10 frames per ns, solid lines and same-color bonding interactions are shown at 5.0 Å and 2.5 Å thresholds (grey, dashed lines).J-K 2D interaction diagram between D 3 R with D 3 R:Gα O βγ and ligands (J) bitopic FOB02-04A Conformation A and (K) bitopic FOB02-04A Conformation B. Specific residues in the binding pocket that interact are shown as sticks and are labelled.Color code for residues and interactions: green, hydrophobic; blue, polar; red, negatively charged; grey, glycine.The solid purple arrow line shows the H-bonding interaction, solid green line shows the π-π-π stacking interaction.L Comparison of FOB02-04A poses obtained from cryo-EM structure with those predicted by molecular docking and molecular dynamic simulations for Conformation A (left panel ) and B (right panel).
of activity and expression of WT mutant D 3 R and G O variants.A-C Tables with summary of pEC 50 values, Emax and expression levels.pEC 50 values and Emax of FOB02-04A, pramipexole and rotigotine binding site on the D 3 R. FOB02-04A, pramipexole and rotigotine are depicted as red, green and cyan sticks.D 3 R is shown as grey cartoons with relevant residues as sticks with carbon colour corresponding to the agonist (red, green and cyan for FOB02-04A, pramipexole and rotigotine respectively).

Frequency
of interactions in D 3 R:Gα O βγ complex H29 1.32 (N2)-E90 2.65 (OE1analysis of TM1 stability in interactions of D 3 R:Gα O βγ with bitopic FOB02-04A (A-C) and pramipexole (D-F).A Closest distances between the carboxyl group of E90 2.65 and the protonated N(ɛ) of H29 1.32 with FOB02-04A.B Frequency of interactions between the same groups in the presence of FOB02-04A.C Closest distances between backbone oxygen atom of E90 2.65 and the protonated N(ɛ) of H29 1.32 with FOB02-04A.D Closest distances between the carboxyl group of E90 2.65 and the protonated N(ɛ) of H291.32  with pramipexole (PDB 7CMU).E Frequency of interactions between the same groups in the presence of pramipexole (PDB 7CMU).F Closest distances between backbone oxygen atom of E90 2.65 and the protonated N(ɛ) of H291.32  with pramipexole (PDB 7CMU).Five independent simulations of D 3 R-Gα O βγ heterotrimer complex are shown, spanning 0.6 μs of cumulative time per system, with the sampling rate of 10 frames per ns, solid lines and same-color shadows representing moving average values and one standard deviation respectively from 50 frames in all cases.Upper and lower boundaries

Frequency
of interactions in D 3 R:Gα O βγ complex

Table 1 .
Cryo-EM data collection, refinement and validation statistics.